DNA mapping takes place using a variety of techniques, which give coarsegrained information on the genome. These techniques are based on sequence specific probes such as in DNA arrays or in fluorescence in-situ hybridization (FISH) with resolution of ˜50 kbp. They can also be based on restriction enzymes that cut the DNA at specific sequences with resolution ˜5 kbp. A common limitation for these techniques is that solely the sequences present in the test can be analysed. Chromosomal banding such as G-banding of metaphase chromosomes circumvents the need for sequence specific probes but with a poor resolution of ˜5 Mbp.
Array comparative genomic hybridisation (CGH) uses DNA arrays to map out the entire genome, specifically looking for copy number variations (CNV). The arrays can be based on BAC clones, cDNA, oligonucleotides or PCR products. The unknown DNA is stained in one colour and mixed with the known DNA stained in another colour. The DNA mixture is allowed to hybridize to the DNA in the array and the result is an array of differently coloured spots. The ratio of the intensities of the two colours in each spot gives information on the CNV. If the intensities of the spots are equal there is no change in copy number. The technique requires hours or days of preparation and several hours to a whole day for the hybridization reactions. Array preparation is complex. Furthermore, the technique is only sensitive to sequences that are represented in the array. It is useful for studies of structural variations that involve a net change in copy numbers. Thus it does not detect balanced translocations and inversions. The resolution is determined by the length and density of probes with a resolution better than 100 kbp. Single-cell array CGH has been demonstrated with a resolution of 1-10 Mbp.
Melting of DNA has been used to detect single basepair variations in genomic DNA using for example constant denaturant gel electrophoresis (CDGE) and denaturing gradient gel electrophoresis (DGGE). Both work well for short stretches of DNA but not for long chromosomal DNA. Scanning electron microscopy has been used to study the melting pattern on a longer length scale [Borovik, A. S.; Kalambet, Y. A.; Lyubchenko, Y. L.; Shitov, V. T.; Golovanov, E. I. Nuc. Acid. Res. 1980, 8, 4165-4184. R. H. Austin, Proc. Natl. Acad. Sci. USA lOl, 10979 (2004)]. This is however a cumbersome and time-consuming technique which requires expensive equipment and specialized training, and it does not lend itself to integration with Lab on a Chip based techniques. It also precludes real-time measurements of melting in aqueous solutions.
Even though there are a large number of ways to analyse DNA, some of them mentioned above, none of the techniques available today present a method wherein it is possible to study the patterns of local AT/GC ratio along large single DNA molecules in an easy, fast and non-expensive way together with good resolution and with a potential of measurements on a single-cell basis.